Diagnosis and follow-up of cystinuria: use of proton magnetic resonance spectroscopy.

Proton Nuclear Magnetic Resonance (NMR) Spectroscopy of urine (as well as of other biological fluids) is a very powerful technique enabling multi-component analysis useful in both diagnosis and follow-up of a wide range of inherited metabolic diseases. Among these pathologies, cystinuria is characterised by accumulation in urine of four dibasic amino acids, namely lysine, arginine, ornithine and cystine; the last one, being only slightly water soluble, generates urolithiasis. The mentioned aminoacids can be detected in the urine NMR spectrum of cystinuric patients, the most abundant being the lysine (5 mM and over are often detected), whose typical signals become very high; arginine and ornithine are also usually detectable, although pathologic concentrations are lower (usually below 2mM). The proposed NMR technique is also suitable in monitoring the therapy with alpha-mercaptopropionylglycine (MPG), providing quantitation of several metabolites of interest in the follow-up of the pathology, like cystine, creatinine and citrate.

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.

Molecular modeling study of the differential ligand-receptor interaction at the mu, delta and kappa opioid receptors.

3D models of the opioid receptors mu, delta and kappa were constructed using BUNDLE, an in-house program to build de novo models of G-protein coupled receptors at the atomic level. Once the three opioid receptors were constructed and before any energy refinement, models were assessed for their compatibility with the results available from point-site mutations carried out on these receptors. In a subsequent step, three selective antagonists to each of three receptors (naltrindole, naltrexone and nor-binaltorphamine) were docked onto each of the three receptors and subsequently energy minimized. The nine resulting complexes were checked for their ability to explain known results of structure-activity studies. Once the models were validated, analysis of the distances between different residues of the receptors and the ligands were computed. This analysis permitted us to identify key residues tentatively involved in direct interaction with the ligand.

New insights into the conformational requirements of B2 bradykinin antagonism.

The conformational profiles of a selected group of a new series of small linear and cyclic penta- and hexapeptides, inspired on the C-terminal segment of second-generation bradykinin (BK) antagonists, were independently computed in order to assess the chemical and geometrical requirements necessary for BK antagonism. Specifically, four cyclic peptides: cyclo-(Gly-Thi-D-Tic-Oic-Arg), cyclo-(Gly-Ala-D-Tic-Oic-Arg), cyclo-(Abu-Ala-Ser-D-Tic-Oic-Arg), cyclo-(Abu-D-Phe-Ala-D-Tic-Oic-Arg), and a linear peptide: Thi-Ser-D-Tic-Oic-Arg were selected for the present study. The first three BK analogs are capable to antagonize kinin-induced rabbit jugular vein and rabbit aorta smooth muscle contraction, while last two show no detectable affinity for the BK B2 receptor. The conformational space of the five peptides was thoroughly explored using simulated annealing (SA) in an iterative fashion as sampling technique. The bioactive conformation was assessed by pairwise cross comparisons between each of the unique low energy conformations found for each of the different peptides studied within a 5 kcal/mol threshold in respect to the global minimum. The conformational profile of the highly potent BK antagonist HOE-140, computed in an independent study, was also used in conjunction with the bioactive form assessed in the present study, to propose a pharmacophore that includes the stereochemical requirements for B2 BK antagonism.

BUNDLE: a program for building the transmembrane domains of G-protein-coupled receptors.

The only information available at present about the structural features of G-protein-coupled receptors (GPCRs) comes from low resolution electron density maps of rhodopsin obtained from electron microscopy studies on 2D crystals. Despite their low resolution, maps can be used to extract information about transmembrane helix relative positions and their tilt. This information, together with a reliable algorithm to assess the residues involved in each of the membrane spanning regions, can be used to construct a 3D model of the transmembrane domains of rhodopsin at atomic resolution. In the present work, we describe an automated procedure applicable to generate such a model and, in general, to construct a 3D model of any given GPCR with the only assumption that it adopts the same helix arrangement as in rhodopsin. The present approach avoids uncertainties associated with other procedures available for constructing models of GPCRs based on a template, since sequence identity among GPCRs of different families in most of the cases is not significant. The steps involved in the construction of the model are: (i) locate the centers of the helices according to the low-resolution electron density map; (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map; (iii) define a local coordinate system for each of the helices; (iv) bring them together in an antiparallel orientation; (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction of the bisector formed between three consecutive helices in the bundle; (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt; and (vii) translate each helix to its center deduced from the projection map.

Conformational analysis of the highly potent bradykinin antagonist Hoe-140 by means of two different computational methods.

The AMBER 4.0 force field was used to perform the characterization of the conformational profile of the highly potent bradykinin antagonist Hoe-140 (D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D-++ +Tic7-Oic8-Arg9). The structural features of the peptide were assessed using two different computational methods, both capable to provide a good sampling of the low-energy conformations of the molecule. Specifically, the conformational space of the peptide was explored: i) computing molecular dynamics trajectories in cycles of high (900 K) and low (300 K) temperature and ii) using simulated annealing (SA) in an iterative fashion. Analysis of the structures characterized indicates that most of the low-energy conformations of the peptide exhibit a betaII'-turn motif at its C-terminus, in agreement with previous experimental and theoretical studies. On the other hand, about a 50% of the low-energy conformations characterized also exhibit different beta-turn type motifs at the N-terminus, whereas the rest of the conformations can be described as bends. Finally, in order to get new insights into the structural requirements necessary to design more potent and selective antagonists of bradykinin, present results were compared with those previously reported by this laboratory on the conformational preferences of the native nonapeptide and its DPhe7 analog.

Conformational study of vasoactive intestinal peptide by computational methods.

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a alpha-helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N-terminal fragment of VIP(1-11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique. In a second step, low-energy structures of the fragment were fused to the remainder of the VIP chain in the form of two noninteracting alpha-helices, according to a model of the structure of the peptide proposed from NMR studies. After investigation for compatibility of each of the low-energy structures of VIP(1-11) with the two helical regions by energy minimization, only 5 of 35 structures were discarded. Analysis of the structures characterized indicates that most of the conformations of VIP(1-11), including the global minimum, can be described as bent conformations. Conformations exhibiting alpha-turns and beta-turns, previously proposed by NMR studies were also characterized. The conformational analysis also suggests that the common structural features found in VIP(1-11) should also be present in VIP. Finally, because of the sequence homology between VIP and Peptide T, and the fact that both are ligands of the CD4 receptor, both sets of low-energy conformations were compared for similarity. The relevance of these results as guidance of the design of new peptide analogs targeted to the CD4 receptor is also discussed.

Diagnosis and follow-up of inborn errors of amino acid metabolism: Use of proton magnetic resonance of biological fluids.

Proton magnetic resonance spectra of biological fluids such as urine, plasma and cerebro-spinal fluid can be used for multi-component analysis of highly concentrated species, thus providing information about the general metabolism of the patient. Hydrogen containing analytes in concentration higher than 10µM are indeed often detectable in biological fluid in 15 minutes by means of an unexpensive 200 MHz spectrometer essentially without sample manipulation. Amino acids, keton bodies, organic acids and other metabolites can be easily estimated by this approach; consequently this technique represents a powerful tool particularly in the diagnosis of inborn errors of amino acid metabolism, when improving the prognosis often depends on a very early diagnosis and on an effective method for monitoring the effects of therapy.In the present paper, several cases of inherited diseases related to amino acid impaired metabolism will be presented to illustrate the importance in the diagnosis. Phenylketonuria, tyrosinemia, cystinuria, ornithinemia, argininosuccinic aciduria, maple syrup urine disease (MSUD), alkaptonuria, lysinuria and other genetic pathologies were in fact unambiguously and rapidly diagnosed by means of the identification in the biological fluids of the relevant accumulating amino acids and/or of their metabolites. The proposed technique is suitable to become, in the future, a useful routine tool for a wide neonatal screening.

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy.

A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.

A physico-chemical approach to the study of the binding interaction between S-adenosyl-L-methionine and polyanions: binding constants and nature of the interaction with sodium poly(styrene sulfonate).

The interaction between S-adenosyl-L-methionine (AdoMet) and sodium poly(styrene sulfonate) NaPSS) was studied by means of ultrafiltration and ultraviolet absorption spectroscopy at several pH values and sodium sulfate concentrations. The results obtained are interpreted mainly in terms of electrostatic interactions and permit the evaluation of the binding constants under different experimental conditions. Furthermore, ultraviolet absorption spectroscopy data show a specific short-range interaction between the aromatic electronic system of AdoMet and the NaPSS aromatic ring. The results indicate that the binding strength is greatly affected by the AdoMet positive charge on the adenine ring. The other positive charges on both the sulfonic pole and the amino acidic group of AdoMet contribute only weakly to the binding to the polyanionic matrix, thus assuring some stability of AdoMet even at physiological pH.

S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B. The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents. The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C. The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

Purification and characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase from human placenta.

5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%. The enzyme has a molecular weight of 98,000, as determined by gel filtration on Sephacryl S-200 and Superose 6B, and is composed by three apparently identical subunits with a molecular weight of 32,500. The isoelectric point is 5.5, and the optimal pH ranges from 7.2 to 7.6. The resistance of the enzyme to thermal inactivation is increased remarkably by the addition of 5'-methylthioadenosine or phosphate. The homogeneous enzyme shows an absolute requirement for -SH-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds. The reaction catalyzed by the enzyme is fully reversible with a Keq of 1.39 X 10(-2) (in the direction of phosphorolysis) at 37 degrees C and pH 7.4. The Km values for 5'-methylthioadenosine, phosphate, adenine, and 5-methylthioribose 1-phosphate are 5, 320, 23, and 8 microM, respectively.

Escherichia coli S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 mumol of 5'-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both S-adenosylhomocysteine and 5'-methylthioadenosine, was partially characterized. The apparent Km for 5'-methylthioadenosine is 0.4 microM, and that for S-adenosylhomocysteine is 4.3 microM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5'-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5'-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5'-methylthioformycin, 5'-chloroformycin, S-formycinylhomocysteine, 5'-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

Studies on the metabolic effects of methylthioformycin.

5'-Methylthioformycin, a structural analog of 5'-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5'-methylthioadenosine and is not metabolized by the cells. 5'-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a Ki value of 2 microM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 microM methylthioformycin. 5'-Methylthioformycin does not exert any significant effect on protein carboxyl-O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.

Inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemic cells by 5'-methylthioadenosine.

5'-Methylthioadenosine is a sulfur-containing nucleoside derived from the metabolism of polyamines which is known to exert an antiproliferative effect on several cell systems in vitro, including the Friend leukemia cell system. We have investigated the role of 5'-methylthioadenosine on the dimethyl sulfoxide-induced differentiation of this system. At a concentration of 400 microM, the drug strongly inhibited (80%) the induced differentiation of Friend cells, and this effect was already observable at a concentration as low as 10 microM (36% inhibition), as evidenced by the benzidine staining procedure and by the dot-blot hybridization of globin mRNA with a human beta-globin probe. Similar results have been obtained by using 5'-S-isobutylthioadenosine, which is a synthetic structural analogue of 5'-methylthioadenosine. The block of differentiation produced by these nucleosides was not mediated by adenine (a catabolite of both molecules) and was not reverted by spermine or spermidine, the two polyamines whose synthesis is inhibited by 5'-methylthioadenosine. We report a decrease of the aminopropyltransferases activities (the enzymes responsible for 5'-methylthioadenosine biosynthesis) in dimethyl sulfoxide-treated Friend cells, which could lead to a decrease of the intracellular content of 5'-methylthioadenosine during the erythroid maturation of Friend cells. The results obtained are consistent with the hypothesis that 5'-methylthioadenosine may act as an endogenous regulator of Friend cell differentiation.

Transport and metabolism of 5'-methylthioadenosine in human erythrocytes.

The transport and metabolism of 5'-deoxy-5'-S-methylthioadenosine have been studied in intact human erythrocytes. The sulfur nucleoside is rapidly accumulated into red cells and the extent of uptake largely exceeds the theoretical equilibrium between inner and outer compartment owing to its conversion into a non-permeable compound, namely 5-methylthioribose 1-phosphate. To characterize the nucleoside transport, phosphate-depleted erythrocytes, in which the methylthioadenosine metabolism is negligible, have been employed. The results indicate that: (i) the transport occurs via a facilitated-diffusion mechanism; (ii) the process is not energy-dependent and (iii) no specific cation is required. The kinetic analyses of both the transport and the metabolism show that the uptake of methylthioadenosine is a result of the tandem action of a transport step of high capacity (Vmax = 604 +/- 51 pmol/10(6) cells per min) and low affinity (Km = 3270 +/- 321 microM) followed by a metabolic step of low capacity (Vmax = 6.6 pmol/10(6) cells per min) and high affinity (Km = 30 microM). Furthermore, a substrate inhibition exerted by methylthioadenosine at high concentration (over 200 microM) on its specific phosphorylase is reported for the first time. Experiments performed with several analogs of the thioether indicate that the adenine amino group and the hydrophobic substituent at the 5'-position are critical for the transport carrier recognition. Adenine is the most powerful inhibitor of methylthioadenosine transport.

Studies on 5'-methylthioadenosine uptake by human erythrocytes.

The transport of 5'-methylthioadenosine across plasma membrane of human erythrocytes occurs by a carrier-mediated mechanism displaying a Km of congruent to 3 mM and a Vmax of congruent to 600 pmol/10(6) cells/min. Phosphate-depleted erythrocytes were employed to distinguish between 5'-methylthioadenosine transport and metabolic trapping. In phosphate medium, where 5'-methylthioadenosine phosphorylase is operative, the uptake of radioactivity increases over the theoretical calculated equilibrium owing principally to conversion of the molecular into 5-methylthioribose-1-phosphate. Newly developed HPLC techniques have been applied to analyze intracellular 5'-methylthioadenosine and its metabolites after exposure of the erythrocytes to [14C]-labelled 5'-methylthioadenosine.